Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device

Abstract

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform-sized EBs, the size of which can be controlled by changing the cross-sectional size of microchannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.

Configurable 2D and 3D spheroid tissue cultures on bioengineered surfaces with acquisition of epithelial–mesenchymal transition characteristics

“…However, the CV values may be optimized and decreased by a redesign of the top channel as well as by the spatial control of uniform side chambers in a microchannel. 11 Importantly, the improved CV in the present study is likely resulted from the deterministic control of equal cell numbers trapped on each perforated membrane, which indicates that this approach may be applied not only for tumor spheroids in cancer studies but also for uniformly sized embryoid bodies in stem cell research.…”

“…Finally, Figure 4d shows multiple spheroids performed on the same microchip at 96 h. Notably, the size of each spheroid was substantially uniform-approximately 78±10 mm in diameter-arranged by through-holes (150 mm pattern diameter). The CV for the spheroids was approximately 13%, whereas that formed in straight channels 11 was approximately 20%.…”

“…5,25,29 Furthermore, the passive method that enables the pumping of multiple fluids using pipettes has been widely adopted, which has the advantages of ease of use, accessibility and a reduction of the complexity of working with pumps and tubing. 11,12,16,37 Here, we successfully adapt these methods in the microchip, which indicates that the microchip could be adopted for use in existing, widely used high-throughput screening instruments, such as liquid-handling robots. 3 This easy, flexible and effective approach may be used for a range of applications in drug discovery, tissue engineering or stem cell research, and should be feasible to integrate with other biological assay components into a monolithic system.…”

“…These surfaces have served as versatile tools to explore fundamental cell biology, tissue engineering and drug development, and thereby became key components in a wide range of applications. 10 Various techniques for patterning cells in 2D or 3D tissue culture have demonstrated the ability to direct cells onto selected areas of a surface, including microcontact printing 8 or soft lithography, 1 microfluidic-or microstructure-based methods, 11,12 active control of cell attachment or suspension by physical forces [13][14][15] and layer-bylayer assembly. 16,17 However, challenges still remain.…”

Flip channel: A microfluidic device for uniform-sized embryoid body formation and differentiation

“…1 For a better recapitulation of ES cell differentiation, the formation of an embryoid body (EB) is pivotal. 2,3 It is well known that microenvironmental factors have a profound impact on directing the differentiation fate of EBs. One of the most important factors is the size and morphology of EBs.…”